The best efforts of a legend of workers have failed to produce an HIV-1 vaccine that is capable of inducing broadly neutralizing antibody. Such antibodies exist and are protective as has been shown by passive transfer with the few monoclonal human antibodies available. While their fine specificity is different these monoclonals frequently demonstrated polyreactivity or self reactivity which may be a key feature of anti-HIV antibodies with broadly neutralizing potential. It has been hypothesized that the processes of immune tolerance normally anergizes or deletes such specificities explaining their absence following vaccination or infection. We have formulated an alternative hypothesis, namely that polyreactive B cells likely producing broadly neutralizing antibody can be either lost directly by somatic hypermutation and selection during the T cell dependent germinal center reaction induced by protein antigens characteristic of HIV vaccines or by failure to compete with more dominant high affinity anti-HIV clones also produced in germinal centers. To evaluate this hypothesis we propose to produce T cell dependent and independent versions of candidate HIV vaccines and test their efficacy at inducing broadly neutralizing anti-HIV antibodies. We will also examine distinct B cell subpopulations to determine their propensity to produce broadly neutralizing antibody and if they may be selectively activated by different molecular forms of HIV vaccine. We will use hybridomas to assess the range of anti-HIV antibodies induced early in the response to HIV vaccine and compare these specificities to ones that dominate in the post germinal center expressed antibody pool. These experiments will be done with human lymphocytes using a huPBL chimera model with markedly improved B cell survival and function recently developed in our laboratory. The goal of this study is to test the hypothesis that an HIV vaccine made by conjugating HIV antigens onto a complex carbohydrate backbone or in the form of a virus like particle has the potential to induce broadly reactive neutralizing anti-HIV antibodies that will inactivate the virus and/or keep it from infecting healthy susceptible cells. The efficacy of this new class of HIV vaccine is derived from its ability to induce responses not altered by somatic mutation and to directly activate subpopulations of protective B cells not normally stimulated by the current HIV vaccine formulations. These protective B cells are evolutionarily adapted to produce rapid responses to both viruses and bacteria and the antibodies they produce following their stimulation frequently show a broad (polyreactive) specificity. It is noteworthy that laboratory selected anti-HIV antibodies with broad neutralizing activity that can protect a host from infection when given in a transfusion show a similar polyreactive character. To determine the effectiveness of this new class of vaccine relative to the current form of vaccine we will immunize humanized mice, which are mice that lack their own immune system that have been reconstituted with human blood lymphocytes. These mice respond to vaccination much like humans would, in that, they make human antibodies for HIV, which can be tested for neutralizing activity in vitro, and the immunized mice can be challenged with infectious virus to demonstrate the in vivo efficacy of the vaccine candidate. Because a number of mice can be reconstituted using blood from a single donor we have the opportunity to compare immune responses to different vaccine formulations in what is immunologically an individual subject thereby eliminating inter-donor response variation. These studies provide a new approach for the design and testing of HIV vaccines.